Genome Mining of Cinnamoyl-Containing Nonribosomal Peptide Gene Clusters Directs the Production of Malacinnamycin.

Cinnamoyl-containing nonribosomal peptides (CCNPs) constitute a unique family of actinobacterial secondary metabolites that display a broad spectrum of biological activities. Here, we present a genome mining approach targeting cyclase and is isomerase to discover new CCNPs, which led to the identification of 207 putative CCNP gene clusters from public bacterial genome databases. After strain prioritization, a novel class of CCNP-type glycopeptides named malacinnamycin was identified. A plausible biosynthetic pathway for malacinnamycin was deduced by bioinformatics analysis.

Evaluation of Viscosity Changes and Rheological Properties of Diutan Gum, Xanthan Gum, and Scleroglucan in Extreme Reservoirs.

The chemically synthesized polymer polyacrylamide (HPAM) has achieved excellent oil displacement in conventional reservoirs, but its oil displacement is poor in extreme reservoir environments. To develop a biopolymer oil flooding agent suitable for extreme reservoir conditions, the viscosity changes and rheological properties of three biopolymers, diutan gum, xanthan gum, and scleroglucan, were studied under extreme reservoir conditions (high salt, high temperature, strong acid, and alkali), and the effects of temperature, mineralization, pH, and other factors on their viscosities and long-term stability were analyzed and compared. The results show that the three biopolymers had the best viscosity-increasing ability at temperatures of 90 °C and below. The viscosity of the three biopolymers was 80.94 mPa·s, 11.57 mPa·s, and 59.83 mPa·s, respectively, when the concentration was 1500 mg/L and the salinity 220 g/L. At the shear rate of 250 s-1, 100 °C~140 °C, scleroglucan had the best viscosification. At 140 °C, the solution viscosity was 19.74 mPa·s, and the retention rate could reach 118.27%. The results of the long-term stability study showed that the solution viscosity of scleroglucan with a mineralization level of 220 mg/L was 89.54% viscosity retention in 40 days, and the diutan gum could be stabilized for 10 days, with the viscosity maintained at 90 mPa·s. All three biopolymers were highly acid- and alkali-resistant, with viscosity variations of less than 15% in the pH3~10 range. Rheological tests showed that the unique double-helix structure of diutan gum and the rigid triple-helix structure of scleroglucan caused them to have better viscoelastic properties than xanthan gum. Therefore, these two biopolymers, diutan gum, and scleroglucan, have the potential for extreme reservoir oil displacement applications. It is recommended to use diutan gum for oil displacement in reservoirs up to 90 °C and scleroglucan for oil displacement in reservoirs between 100 °C and 140 °C.

Simulation Study on Molecular Behavior of Rhamnolipids and Biobased Zwitterionic Surfactants at the Oil/Water Interface: Effect of Rhamnose Moiety Structures.

Molecular behavior of rhamnolipid mixed with a biobased zwitterionic surfactant at an n-hexadecane/water interface has been studied, and the effects of a rhamnose moiety and composition are evaluated. Results showed that rhamnolipid abundantly interacts with biobased surfactant EAB by means of hydrophobic interactions between aliphatic tails and electrostatic interactions between headgroups, including the attractive interaction between COO- of rhamnolipids and N+ of biobased surfactants and the repulsive interaction between COO- of both surfactants. Dirhamnolipid has a larger number of bound Na+ and a more stable bound structure of COO- ∼ Na+, which screens the repulsive interaction between two kinds of surfactants and shows a more homogeneous distribution with biobased surfactants. The interfacial tension between n-hexadecane and water has been synergistically reduced by dirhamnolipids mixed with biobased surfactants at a higher molar ratio of biobased surfactants. Monorhamnolipids show a strengthened interaction with N+ of biobased surfactants and a more stable hydrogen bond with water relative to that of dirhamnolipids, and there is no synergistic effect in lowering the interfacial tension for the mixture of monorhamnolipids and biobased surfactants. The present work provides details of the molecular behavior of biosurfactant rhamnolipids mixed with biobased surfactants and obtains the key factor in affecting the interfacial properties of the binary system.

Improving Rhamnolipids Biosynthesis in Pseudomonas sp. L01 through Atmospheric and Room-Temperature Plasma (ARTP) Mutagenesis.

Biosurfactants have significant applications in various industries, including microbial-enhanced oil recovery (MEOR). While the state-of-the-art genetic approaches can generate high-yield strains for biosurfactant production in fermenters, there remains a critical challenge in enhancing biosurfactant-producing strains for use in natural environments with minimal ecological risks. The objectives of this work are enhancing the strain's capacity for rhamnolipids production and exploring the genetic mechanisms for its improvement. In this study, we employed atmospheric and room-temperature plasma (ARTP) mutagenesis to enhance the biosynthesis of rhamnolipids in Pseudomonas sp. L01, a biosurfactant-producing strain isolated from petroleum-contaminated soil. Following ARTP treatment, we identified 13 high-yield mutants, with the highest yield of 3.45 ± 0.09 g/L, representing a 2.7-fold increase compared to the parent strain. To determine the genetic mechanisms behind the enhanced rhamnolipids biosynthesis, we sequenced the genomes of the strain L01 and five high-yield mutants. A comparative genomic analysis suggested that mutations in genes related to the synthesis of lipopolysaccharides (LPS) and the transport of rhamnolipids may contribute to the improved biosynthesis. To the best of our knowledge, this is the first instance of utilizing the ARTP approach to improve rhamnolipid production in Pseudomonas strains. Our study provides valuable insights into the enhancement of biosurfactant-producing strains and the regulatory mechanisms of rhamnolipids biosynthesis.

Degradation of crude oil in a co-culture system of Bacillus subtilis and Pseudomonas aeruginosa.

Microbial remediation has been regarded as one of the most promising decontamination techniques for crude oil pollution. However, there are few studies on the interaction of bacteria in the microbial community during bioremediation. The aim of this work was to research the promotion of defined co-culture of Bacillus subtilis SL and Pseudomonas aeruginosa WJ-1 for biodegradation of crude oil. After 7 days of incubation, the analysis of residual oil, saturated and aromatic fraction in the samples showed that the degradation efficiency of them was significantly improved. The degradation efficiency of crude oil was enhanced from 32.61% and 54.35% in individual culture to 63.05% by the defined co-culture of strains SL and WJ-1. Furthermore, it was found that the defined co-culture system represented relatively excellent performance in bacterial growth, cell surface hydrophobicity (CSH) and emulsification activity. These results indicated that the combination of Bacillus subtilis and Pseudomonas aeruginosa can effectively promote the degradation and utilization of crude oil, which may provide a new idea for the improvement of bioremediation strategies. GRAPHICAL ABSTRACT.

Research advances of microbial enhanced oil recovery.

Microbial enhanced oil recovery (MEOR), characterized with the virtues of low cost and environmental protection, reflects the prevalent belief in environmental protection, and is attracting the attention of more researchers. Nonetheless, with the prolonged slump in global oil prices, how to further reduce the cost of MEOR has become a key factor in its development. This paper described the recent development of MEOR technology in terms of mechanisms, mathematical models, and field application, meanwhile the novel technologies of MEOR such as genetically engineered microbial enhanced oil recovery (GEMEOR) and enzyme enhanced oil recovery (EEOR) were introduced. The paper proposed three possible methods to decrease the cost of MEOR: using inexpensive nutrients as substrates, applying a mixture of chemical and biological agents, and utilizing crude microbial products. Additionally, in order to reduce the uncertainty in the practical application of MEOR technology, it is essential to refine the reservoir screening criteria and establish a sound mathematical model of MEOR. Eventually, the paper proposes to combine genetic engineering technology and microbial hybrid culture technology to build a microbial consortium with excellent oil displacement efficiency and better environmental adaptability. This may be a vital part of the future research on MEOR technology, which will play a major role in improving its economic efficiency and practicality.

Single-Cell-Based High-Throughput Cultivation and Functional Characterization of Biosurfactant-Producing Bacteria from Soil and Oilfield-Produced Water.

Biosurfactants are a group of surface-active compounds that can be produced by diverse microorganisms. They have been widely used in various industrial fields. Reducing production costs, improving efficiency, and collecting more diverse producing strains have become major challenges in the biosurfactant industry. These challenges could be overcome by screening for more diverse and efficient biosurfactant-producing strains. The conventional methods for the isolation and functional characterization of microorganisms are laborious and biased toward fast-growing or strongly competitive microorganisms. Here, we established a high-throughput approach of single-cell-based cultivation and functional characterization of biosurfactant-producing bacteria (SCCBB). This approach combines single-cell cultivation with the detection of optical distortions. Using this approach, we isolated 431 strains with biosurfactant production potential from petroleum-contaminated soil and oilfield-produced water. The surfactant production capabilities of the strains were subsequently validated using surface tension measurements, TLC, and CMC measurements. To investigate the industrial production potential, we optimized the production conditions of a representative glycolipids-producing strain, Pseudomonas sp. L01, using response surface methodology (RSM). Optimal conditions yielded a crude biosurfactant yield of 8.43 g/L in a flask. Our work provides a high-throughput approach to the isolation and screening of biosurfactant-producing bacteria, as well as other functional bacteria in a wide range of fields.

Study on the thermal washing of oily sludge used by rhamnolipid/sophorolipid binary mixed bio-surfactant systems.

Demulsification and crude oil desorption are usually a necessary step for the treatment of oily sludge in the petroleum industry. In this study a binary mixed bio-surfactant (rhamnolipid / sophorolipid, RL/SL) was used to strengthen the removing oil efficiency for oily sludge by thermal washing method. Surface tension values of the single and the mixed surfactants were carried out to investigate the effect of mixing systems on reducing critical micelle concentrations (CMC) value. The models proposed by Clint, Rubingh and Gibbs et al. had been employed to interpret the formation of mixed micelles and synergism and found out in case of the mass ratios of 4:6 the synergism was the strongest in RL and SL mixed surfactant systems, which was selected as the washing agents to treat the oily sludge produced from Huabei oilfield. Through the optimization of oil washing process parameters, the oil removal rate reached the maximum value (95.66%, residual oil rate 1.98%) at the condition of heating temperature of 45 °C, detergents concentration of 500 mg/L, washing time of 3 h, liquid/solid mass ratio of 1:4, stirring speed of 300 r/min, and washing 4 times. The factors affecting the oil washing effect were analyzed from the composition and performance characteristics of oily sludge samples, washing oil system and washing process parameters. The results showed that low oil content of oily sludge, small specific surface area, strong wetting and solubilization of the oil-washing system all can increase the oil-washing effect and the washing time and temperature had a great influence on the oil-washing effect. Compared with the results of other researchers, the oil washing temperature and the concentration of oil washing agent were significantly lower and high oil removal rate and low residual oil rate were obtained in this study. It was confirmed that thermal oil washing method using RT/SL binary bio-surfactant mixing system was proved to a high-efficiency, low-consumption and wide range of applications technology.

Biosurfactant production by Bacillus subtilis SL and its potential for enhanced oil recovery in low permeability reservoirs.

Microbial enhanced oil recovery (MEOR) technology is an environmental-friendly EOR method that utilizes the microorganisms and their metabolites to recover the crude oil from reservoirs. This study aims to research the potential application of strain SL in low permeability reservoirs. Strain SL is identified as Bacillus subtilis by molecular methods. Based on the mass spectrometry, the biosurfactant produced by strain SL is characterized as lipopeptide, and the molecular weight of surfactin is 1044, 1058, 1072, 1084 Da. Strain SL produces 1320 mg/L of biosurfactant with sucrose as the sole carbon source after 72 h. With the production of biosurfactant, the surface tension of cell-free broth considerably decreases to 25.65 ± 0.64 mN/m and the interfacial tension against crude oil reaches 0.95 ± 0.22 mN/m. The biosurfactant exhibits excellent emulsification with crude oil, kerosene, octane and hexadecane. In addition, the biosurfactant possesses splendid surface activity at pH 5.0-12.0 and NaCl concentration of 10.0% (w/v), even at high temperature of 120 °C. The fermentation solution of strain SL is applied in core flooding experiments under reservoir conditions and obtains additional 5.66% of crude oil. Hence, the presented strain has tremendous potential for enhancing the oil recovery from low-permeability reservoirs.

Study on the treatment of oily sludge in oil fields with lipopeptide/sophorolipid complex bio-surfactant.

A systematic study had been carried out to get insight into the micellar behavior of anionic lipopeptide (LT) and nonionic sophorolipid (SL) in their different mass ratio mixed state using the technique of tensiometry. The models proposed by Clint, Rubingh and Gibbs et al. had been employed to interpret the formation of mixed micelles and found out synergism. The obtained experimental critical micelle concentrations (CMC) were lower than the ideal CMCs, indicating negative deviation from ideal behavior for all multi-component mixed micelles formation. A suited binary bio-surfactant mixing system was selected as the washing agents to treat the oily sludge produced from Huabei oilfield by the thermal bio-surfactant washing method. The results showed that in case of the mass ratios of 8:2 the CMC was dramatically decreased and synergism was the strongest in LT and SL bi mixed surfactant systems. The studied binary mixed bio-surfactant system showed higher washing efficiency for oily sludge than single surfactant system. In addition, the washing power of binary mixed bio-surfactants towards oily sludge was the best at below washing conditions: (a) the concentration of the mixed system (100 mg/L), (b) temperature (55 â„ƒ), (c) ratio of sludge/liquid (1:3), (d) washing time (3 h), and (e) stirring speed (300 rpm). Certainly, the washing abilities of the selected surfactants not only depend on their mixing ratio and washing conditions but also associate with microstructure and mineral components of oily sludge.

Tepidiphilus olei sp. nov., isolated from the production water of a water-flooded oil reservoir in PR China.

A novel, moderately thermophilic, Gram-stain-negative bacterium, designated strain J18T, was isolated from a water-flooded oil reservoir. Cells were aerobic, oxidase- and catalase-positive, with a polar flagellum. Growth occurred at 35-60 °C and at pH 6-8.5. The respiratory quinones were ubiquinone 8 and ubiquinone 9. The dominant cellular fatty acids were C16 : 0, C17 : 0 cyclo, C19 : 0 cyclo ω8c and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c). The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, an unidentified aminolipid, an unidentified phospholipid and an unidentified aminophospholipid. The strain showed the highest 16S rRNA gene sequence similarities to Tepidiphilus margaritifer DSM 15129T (98.6 %), Tepidiphilus succinatimandens DSM 15512T (98.4 %) and Tepidiphilus thermophilus DSM 27220T (98.1 %), respectively, and the similarity to other species was lower than 93 %. In the phylogenetic trees, it constituted a unique sub-cluster within the genus Tepidiphilus. The DNA G+C content of strain J18T was 64.44 mol%. As compared with the type strains, the genome-to-genome distances of strain J18T were 34.7-40 %. These results confirmed the separate species status of J18T with its close relatives. On the basis of physiological, chemotaxonomic and phylogenetic analyses along with the low levels of identity at the whole-genome level, it can be concluded that strain J18T represents a new species of the genus Tepidiphilus, for which the name Tepidiphilus olei sp. nov. is proposed. The type strain of T. olei is J18T (=CGMCC 1.16800T=LMG 31400T).

[Effect of N2O produced by indigenous denitrifiers in oil reservoir on the physical properties of crude oil]. / 油藏本源反硝化功能菌的产气作用(N2O)及对原油物性的影响.

The success of microbial enhanced oil recovery (MEOR) relies on complex microbial processes. Nevertheless, the contribution and mechanism of in-situ denitrification to microbial oil recovery remain unclear. In this study, eight denitrifying bacterial strains, designated T1, D1, D44, D46, D15, S1, S2 and S6, were isolated from the produced water of Xinjiang Oilfield, China, by a double layered plate method. The16S rDNA gene sequences of these denitrifying strains shared 100% similarity with Pseudomonas stutzeri (T1, D1, and D44), Pseudomonas putida (D46 and D15), and Pseudomonas aeruginosa (S1, S2, S6), respectively. The N2O production effects of these strains on the physical properties of crude oil were evaluated with batch experiment. Results showed that the highest total gas yield was observed with sucrose as carbon source, and the maximal concentration of N2O occurred with glycerol as carbon source. The denitrification process by these bacterial strains led to volume expansion and viscosity reduction of crude oil. Crude oil expansion rate was positively correlated with the concentration of N2O, with a correlation coefficient of 0.983, but not correlated with the volume of total gas production. Strain S1, S2, and S6 produced 530-730 mg·L-1 of surfactant using glycerol as ole carbon source, which could reduce surface tension and emulsify crude oil. However, these surfactant-producing strains produced less N2O, exhibited weaker effects on oil swelling and viscosity reduction, compared to the none-surfactant-producing denitrifying strains. Our results suggested that more attention should be paid to the ability of N2O production by denitrifying bacteria when exploiting microbial resources towards enhancing oil recovery.

Characterizing the microbiome in petroleum reservoir flooded by different water sources.

Petroleum reservoir is an unusual subsurface biosphere, where indigenous microbes lived and evolved for million years. However, continual water injection changed the situation by introduction of new electron acceptors, donors and exogenous microbes. In this study, 16S-rRNA gene sequencing, comparative metagenomics and genomic bins reconstruction were employed to investigate the microbial community and metabolic potential in three typical water-flooded blocks of the Shen84 oil reservoir in Liaohe oil field, China. The results showed significant difference of microbial community compositions and metabolic characteristics existed between the injected water and the produced water/oil mixtures; however, there was considerable uniformity between the produced samples in different blocks. Microbial communities in the produced fluids were dominated by exogenous facultative microbes such as Pseudomonas and Thauera members from Proteobacteria phylum. Metabolic potentials for O2-dependent hydrocarbon degradation, dissimilarly nitrate reduction, and thiosulfate­sulfur oxidation were much more abundant, whereas genes involved in dissimilatory sulfate reduction, anaerobic hydrocarbon degradation and methanogenesis were less abundant in the oil reservoir. Statistical analysis indicated the water composition had an obvious influence on microbial community composition and metabolic potential. The water-flooding process accompanied with introduction of nitrate or nitrite, and dissolved oxygen promoted the alteration of microbiome in oil reservoir from slow-growing anaerobic indigenous microbes (such as Thermotoga, Clostridia, and Syntrophobacter) to fast-growing opportunists as Beta- and Gama- Proteobacteria. The findings of this study shed light on the microbial ecology change in water flooded petroleum reservoir.

Exopolysaccharide production by an indigenous isolate Pseudomonas stutzeri XP1 and its application potential in enhanced oil recovery.

In this study, a rapid and efficient method for screening biopolymer producers was established using 96-well plates. An indigenous biopolymer producer Pseudomonas stutzeri XP1 was isolated from Xinjiang oil reservoirs, China. Strain XP1 can grow and produce 16 g/l biopolymer using corn starch and nitrate. Produced biopolymer increased culture viscosity up to 2384 mPa s. Biopolymer showed rheological properties and pseudo-plastic behavior. The viscosity of 8 g/l biopolymer solution kept higher than 25 mPa s at 20-50 °C and pH values (5-9) and increased to 7600 mPa s with NaCl concentrations increasing to 2%. Gel permeation chromatography data showed that the biopolymer average molecular mass was 1.65 × 106 Da. Gas chromatography revealed that the monosaccharide composition in biopolymer was glucose. Core flooding experiments revealed that extra 13.56% of oil was recovered by in situ biopolymer production of strain XP1. Properties of strain XP1 and the biopolymer produced make them promising for enhanced oil recovery.